Toll-like Receptor 2 Polymorphisms Impose Considerable Impacts On Acute Myelocytic Leukemi Occurrence

Background: This research aimed to explore the genetic association of Toll-like receptor 2 (TLR2) gene polymorphisms with acute myelocytic leukemia (AML) susceptibility in Chinese population. Methods: Firstly, the genotypes of TLR2 polymorphism were detected in 148 AML patients and 126 healthy controls by polymerase chain reaction (PCR). The genotype distribution of every polymorphism in the control group was detected whether conformed to Hardy-Weinberg equilibrium (HWE). The genotype frequency difference of TLR2 polymorphism between the case and control groups was compared by chi-square test and odds ratio (OR) with 95% condence interval (95%CI) was calculated to express the risk of AML resulted from genetic variant of TLR2. Results: For TLR2 rs3804099 polymorphism, the heterozygous genotype TC was detected the signicantly lower frequency in AML patients than that in the controls (P=0.031), so was C allele (P=0.021), which showed that rs3804099 was associated with the decreased the risk of AML (OR=0.580, 95%CI=0.354-0.953; OR=0.641, 95%CI=0.438-0.938). Similarly, GG genotype and G allele of rs1898830 also showed the obviously association with the risk reduction of AML (GG vs. AA: OR=0.430, 95%CI=0.200-0.924, G vs. A: OR=0.644, 95%CI=0.451-0.919). However, no signicant association was found in rs7656411 with AML. Conclusions: TLR2 rs3804099 and rs1898830 polymorphisms may be the protective factors for AML, but not rs7656411.

Conclusions: TLR2 rs3804099 and rs1898830 polymorphisms may be the protective factors for AML, but not rs7656411.

Background
Acute myelocytic leukemia (AML) is a kind of progressive malignant tumor in hematopoietic system with low cure rate and high recurrence rate [1,2]. The pathological features of AML are rapidly increasing abnormal leucocytes aggregate in the bone marrow to disturb the production of normal cells such as erythrocytes and platelets [3]. In general, it easily attacks the adults, but 15% of childhood leukemia cases are explained by AML, too [4]. If not timely treatment, AML is fatal in a few months, which brings about huge economic burden and life pressures. The pathogenesis of AML is multifactorial result consisted of environmental and genetic factors [5,6]. The known environmental factors include smoking, obesity, X radiation and chemical carcinogens [7,8], however, only a small part of people suffer from AML which expose to risk environmental factors. Therefore, genetic factors play a key role in the onset of AML.
Nowadays, identi ed genes associated with AML are few not to explain the etiology.
Toll-like receptors (TLRs) is a set of conserved pattern recognition receptors which act on innate immune system against pathogenic microorganisms [9]. They are detected the expression in immune cells, including neutrophils, monocytes, dendritic cells, macrophages, T cells, B cells and NK cells [10]. TLRs can in uence the secretion of cytokines and chemokines to participate in the immune activity [11,12].
What's more, the up-regulated expression of TLRs is also detected in several cancers and tumor cell lines [13,14]. TLR2 is an important member of TLRs family with the broadest expression in human cells and recognizes the most types of pathogenic microorganisms. The activation of TLR2 signaling has been found to induce the activation of mitogen-activated protein kinase (MAPK) and NF-κB so as to prolong the survival time of tumor cells [15]. So far, the researches about the association of TLR2 with AML are few.
TLR2 coding gene TLR2 is located on chromosome 4q31.3 [16] and had been identi ed a number of single nucleotide polymorphisms (SNPs). In the present study, we selected three common SNPs in the exon, intron and 3'UTR of TLR2 to investigate the role in AML occurrence risk in a Chinese Han population.

Cases and controls
The current study included 148 AML patients and 126 healthy controls as the case and control groups, respectively. In the case group, patients with AML were inpatients from haemal internal medicine of PanYu Central Hospital, including 83 males and 65 females. They were diagnosed based on morphological and phenotypic data according to the criteria of World Health Organization (WHO) [17].
Their age range was from 17 to 69 years old. At the same time, the healthy controls were selected experienced the physical examination in the same hospital with the cases and consisted of 64 males and 62 females with the age of 23-76. The healthy controls were frequency-matched with AML patients in age and sex. All subjects were all Chinese Han population and they were no blood relationship. This study design was reviewed and supported by the Research Ethics Committee of PanYu Central Hospital, meanwhile, the objective and program were informed all subjects before collecting blood samples. Moreover, written consents were signed by every subject. DNA extraction 2ml peripheral venous blood were collected from every subject with empty stomach on the early morning and placed in the blood collection tube with EDTA2Na, -80℃. Whole blood genomic DNA was extracted using TIANamp Genomic DNA Kit according to the manufacturer's instruction and stored at -20℃ for standby application.
The genotyping of TLR2 polymorphisms The genotyping of TLR2 rs3804099, rs1898830 and rs7656411 polymorphisms was conducted by polymerase chain reaction (PCR) with direct sequencing. First of all, PCR primers were designed by Primer Premier 5.0 software and synthesized in Shanghai Sangon Biotech Co., Ltd. The relative information of primer sequences was showed in Table 1. PCR system was a volume of 25.0μl mixture, including 12.5μl PCR Mix, 2.0μl DNA template, each 1.0μl of forward and reverse primers and nally added ddH 2 O to 25.0μl. PCR procedure was conducted according to the following steps: pre-degeneration at 95℃ for 3min, 33 cycles of degeneration at 95℃ for 45s, annealing at 56℃ (rs1898830) and 60℃ (rs3804099 and rs7656411) for 30s, extension at 72℃ for 30s, and nal extension at 72℃ for 7min. The quality of PCR products was tested by agarose gel electrophoresis.
The eligible PCR products were sent to Sangon Biotech (Sangon, Shanghai) for sequencing so as to determine the genotype of every TLR2 polymorphism in all subjects.

Statistical analysis
In this study, the genotype frequency was obtained using direct counting and the genotype distribution of every polymorphism in the control group was detected by χ² test whether was consistent with Hardy-Weinberg equilibrium (HWE). The genotype, allele frequency difference of TLR2 polymorphisms between AML patients and the healthy controls were calculated by chi-square test. Odds ratio (OR) and 95% con dence interval (95%CI) was used to represent the relative risk of AML caused by TLR2 variants. All data were managed by PASW Statistics 18.0 software and showed in the form of ±s and %. case and control groups (24.9 ± 3.4 and 23.8 ± 2.6, P = 0.000). 37.84% of AML patients were smokers and the percentage was 29.37% in controls, which suggested smoking was not a in uence factor of AML in this population (P = 0.140). Differently, exposed to chemical carcinogens was a risk factor for AML occurrence (P = 0.024). The status of HWE was checked based on the genotype distribution of polymorphism in the control group, the results were showed in Table 3. The genotype distribution of TLR2 rs3804099, rs1898830 and rs7656411 in the control group were all consistent with HWE requirement (P=0.161, 0.466 and 0.245 respectively), indicating our study population was a representative group.
The association analysis of TLR2 polymorphisms with AML risk The genotype and allele frequencies of TLR2 polymorphisms were calculated and displayed in Table3.
We can see that the genotype and allele distributions of rs3804099 and rs1898830 between the case and control groups were very different, but not rs7656411.
Firstly, the heterozygous genotype TC of rs3804099 was signi cantly lower frequency in AML patients than that in the controls (P=0.031), compared with the common genotype TT and we also obtained the similar results in allele (P=0.021

Discussion
The occurrence of AML is caused by karyotype mutation of pluripotent stem cells and lightly differentiated precursor cells [18]. Abnormal chromosome karyotype, especially chromosome translocation is typical cytogenetic characteristic of AML, or else, apparent molecular abnormalities exist in the pathogenesis of AML. The onset of AML is in all ages and it is most common in the elderly, with poor prognosis. Exact explaining the pathology of AML is a urgent issue. AML is a complex multifactorial disease described by precious studies. On the one hand, a series of environmental factors have identi ed, mainly referring to radiation [19], chemical factor and infection. On the other hand, genetic factors play the key role in the occurrence of AML. So far, multiple gene variants are found to be associated with AML, including FLT3-ITD, IDH1, IDH2, DNMT3A NPM1 and so on. However, these researches are far away from revealing the etiology of AML.
TLR is one of innate immune receptors and play an important role in activation and regulation of innate immunity, and the induction of adaptive immunity. Meanwhile, it also has an in uence on cardiovascular disease, autoimmunity development and tumors [20,21]. They involve in maintaining the effective immune response in the acute infection and injury. TLR signaling is necessary for normal immune reaction, however, studies have been reported that the abnormality of TLR signaling is related to ineffective hematopoiesis and hematopoietic malignancy [22,23]. TLR family includes 10 members, of which TLR2 is the broadest one in expression and it also identi es the most pathogenic microorganisms.
In previous report, the high expression of TLR2 is found in AML patients and AML patients with high expression of TLR2 have the shorter survival time than that patients with low expression [11]. So, TLR2 may involve in the etiology of AML.
Its encoding TLR2 is consisted of 5 exons and introns and has been identi ed a number of SNPs. TLR2 polymorphisms can in uence host innate immune response and immune defence through regulating transcription activity or changing protein structure. Multiple SNPs in TLR2 are studied the association with some diseases. Rs3804099 is a synonymous mutation in exon3 of TLR2 with the substitution of T/C and it has been found to be associated with several diseases, such as in ammatory disease, cardiovascular disease, immune disease and cancer [24]. Rs1898830 is a mutation of A/G in intron1 region of TLR2 and the carriage of AA induces the expression of target genes in IKK-β and NF-κB pathway and promotes the secretion of pro-in ammatory factor, including interleukin and chemokine, such as TNF, IL-6, CCL3 and CCL2 [25,26]. It also participates in many diseases pathogenesis, certainly including cancer [27]. For rs7656411 is a SNP in 3' untranslated region (3'UTR) of TLR2 with a base mutation of G/T and it may affect the expression of TLR2 mRNA.
In the present study, we explored the genetic association of these three common SNPs in TLR2 with AML susceptibility based on a Chinese Han population. In this study population, smoking was not a risk factor of AML, but obesity and exposed to chemical carcinogens were signi cantly correlated to the occurrence of AML. Rs3804099 was revealed to be related to the development of AML and the carriage of TC genotype and C allele could signi cantly decreased the susceptibility of people to AML. Similar results were showed in rs1898830, that is people carrying GG genotype and G allele of rs1898830 had the lower risk suffering from AML, compared with AA genotype and A allele respectively. However, rs7656411 was not checked the obvious association with AML in our study group. This is the rst time to investigate the role of TLR2 multiple SNPs in the risk of AML in Chinese population.

Conclusions
In conclusion, TLR2 may play an important role in AML pathogenesis and the genetic variants may cause the expression change of TLR2 to contribute the risk to disease, including AML. We have identi ed rs3804099 and rs1898830 in TLR2 associated with AML occurrence based in the current study. But some limitations in uence the nal results, too and most of them are small samples, single group and environmental factors. Therefore, more further researches are needed to verify this results with welldesign and explore the detailed mechanism in the future.

List Of Abbreviations
Toll The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.